Emerging viruses that jump from one host to another to cause epidemics of disease pose serious risks to human health and wellbeing. Similar events occur in animals to create epidemic or pandemic pathogens with new host ranges, which can provide important and highly informative models to reveal the fundamental processes that underpin disease emergence. This is the renewal application for a project that will continue to combine the laboratory studies undertaken by Colin Parrish at Cornell University and the comparative genomic work of Eddie Holmes at the University of Sydney. We have worked closely together for the past 12 years in a productive collaboration that has brought our complementary skills to bear on studies of viral evolution associated with host jumping. Through analysis of virus structures, functions, and host interactions we have identified some of the fundamental biological processes that determine disease emergence. Here, we will continue and extend these studies by utilizing three model systems in which viruses have jumped hosts and then spread epidemically: the emergence of the H3N8 and H3N2 canine influenza viruses (CIV) and that of canine parvovirus (CPV). The results of this research program will be broadly applicable to understanding the emergence of viruses and potentially that of other pathogens. We have completed the main aims of the previous award period. We will now build on and extend those results by defining key steps in the emergence of viruses in new hosts, using these naturally-derived model viruses in various combinations to carry out the following three Aims: Aim 1. The repeatability of virus emergence. A central aspect of our proposal is determining the likely repeatability of virus adaptation to new hosts, particularly whether strong functional constraints lead to a convergence of both the underlying mutations (genotype) and host range (phenotype). To test these ideas we will analyze the adaptation of CIV and CPV to their different hosts, of CIV and CPV as they evolve in nature, and determine the connection between virus evolution and receptor variation. Aim 2. Understanding virus-host receptor interactions and their evolution. For both CIV and CPV we will define the fine-structure mechanisms of virus-host cell interactions, including analysis of the biophysical interplay between viral proteins and their ligands, and the alternative pathways available. This will allow us to define, for the first time, the intricate virus-host interactions that underpin emergence. Aim 3. Determining the rates and patterns of genetic variation in ssDNA viruses. Single-strand (ss) DNA viruses like parvoviruses are replicated by host cell DNA polymerases which show high fidelity when replicating host DNA, but which allow variation of ssDNA viruses, resulting in RNA-like evolutionary rates. We will determine the mutation rates in the parvoviruses, the patterns of sequence variation arising in experimental systems, and the role of DNA sequence context in the emergence of specific viral mutations.